JP5738756B2 - ファミリー44のキシログルカナーゼの変異体を含む洗剤組成物 - Google Patents
ファミリー44のキシログルカナーゼの変異体を含む洗剤組成物 Download PDFInfo
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- JP5738756B2 JP5738756B2 JP2011512553A JP2011512553A JP5738756B2 JP 5738756 B2 JP5738756 B2 JP 5738756B2 JP 2011512553 A JP2011512553 A JP 2011512553A JP 2011512553 A JP2011512553 A JP 2011512553A JP 5738756 B2 JP5738756 B2 JP 5738756B2
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38681—Chemically modified or immobilised enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Detergent Compositions (AREA)
- Enzymes And Modification Thereof (AREA)
Description
i)この位置を占めるアミノ酸の下流へのアミノ酸の挿入、
ii)この位置を占めるアミノ酸の欠失、又は
iii)この位置を占めるアミノ酸の異なるアミノ酸での置換、であり、
親キシログルカナーゼはファミリー44のキシログルカナーゼであり、変異体はキシログルカナーゼ活性を有する。
キシログルカナーゼ活性:用語「キシログルカナーゼ活性」は、本明細書においては、キシログルカンの酵素触媒による加水分解として定義される。この反応は、キシログルカンの1,4−β−D−グルコース結合のエンド加水分解を伴う。本発明の目的上、キシログルカナーゼ活性は、AZCL−キシログルカン(Megazymeより入手)を反応基質として使用して決定される。アッセイは、本出願の実施例2に記載のような、又はWO 01/62903に記載のような様々な方法で実施され得る。キシログルカナーゼ活性の一単位(XyloU)は、WO 01/62903の60頁、3〜17行目に記載のアッセイ方法を参照することにより定義される。
(同一残基×100)/(アラインメントの長さ−アラインメント中のギャップの合計数)
(同一デオキシリボヌクレオチド×100)/(アラインメントの長さ−アラインメント中のギャップの合計数)。
本発明の目的上、配列番号3に開示されるキシログルカナーゼのアミノ酸配列は、別のキシログルカナーゼ中の対応するアミノ酸残基を決定するのに使用される。別のキシログルカナーゼのアミノ酸残基は、配列番号3に開示されるキシログルカナーゼのアミノ酸配列とアラインメントされ、このアラインメントに基づいて、配列番号3に開示されるキシログルカナーゼのアミノ酸配列中の任意のアミノ酸残基に対応するアミノ酸の位置番号を決定することができる。
本発明において、親キシログルカナーゼは、(a)ファミリー44のキシログルカナーゼとも呼ばれる、グルコシル加水分解酵素のファミリー44に属するキシログルカナーゼ;若しくは(b)配列番号3、配列番号5、及び配列番号7からなる群から選択されるポリペプチド;又は(c)配列番号3の成熟したポリペプチドと少なくとも75%同一性を有するアミノ酸配列を含むポリペプチド;あるいは(d)少なくとも中度のストリンジェンシー条件下で、(i)配列番号1、配列番号4、又は配列番号6の成熟したポリペプチドコード配列、(ii)配列番号1、配列番号4、又は配列番号6の成熟したポリペプチドコード配列を含むゲノムDNA配列、又は(iii)(i)若しくは(ii)の完全長の相補鎖、とハイブリダイズする、ポリヌクレオチドによってコードされるポリペプチド;又は(e)配列番号1の成熟したポリペプチドコード配列と少なくとも70%同一性を有するヌクレオチド配列を含むポリヌクレオチドによってコードされるポリペプチド;のいずれかである。
親キシログルカナーゼの変異体は、ランダムな及び/又は部位特異的な突然変異誘発、合成遺伝子構築、半合成遺伝子構築、ランダム変異導入法、シャッフル等などの、当該技術分野において既知である任意の突然変異誘発手段に従って調製することができる。
本発明において、親キシログルカナーゼの単離された変異体は、位置番号68、123、156、118、200、129、137、193、92、83、149、34、340、332、9、76、331、310、324、498、395、366、1、374、7、140、8、14、21、211、37、45、13、78、87、436、101、104、111、306、117、119、414、139、268、142、159、164、102、168、176、180、482、183、202、206、217、4、222、19、224、228、232、2、240、244、5、247、249、328、252、259、406、267、269、275、179、166、278、281、288、298、301、18、302、165、80、303、316、169、322、120、146、342、348、147、353、380、468、382、383、38、384、389、391、10、392、396、177、397、399、409、237、413、253、415、418、40、443、445、148、449、225、450、454、3、455、456、299、461、470、204、476、488、347、及び507からなる群から選択される1つ以上(いくつか)における改変を含み、ここで、キシログルカナーゼ活性を有する変異体は、親キシログルカナーゼのアミノ酸配列との同一性の度合が少なくとも70%、より好ましくは少なくとも75%、より好ましくは少なくとも80%、より好ましくは少なくとも85%、更により好ましくは少なくとも90%、より好ましくは少なくとも95%、より好ましくは少なくとも約97%、最も好ましくは少なくとも98%、及び更により好ましくは99%であるアミノ酸配列を含む。位置の番号付けは、配列番号3のアミノ酸配列に関連している。好ましくは、上記で特定した位置の1つ以上における改変を含む変異体は、親キシログルカナーゼと比べて、洗剤、好ましくは液体洗剤中での増加した安定性を有する。
V1*+V2*+H3*;
V1Q+*1aE+*1bV;
H3A;
H3A+H436A;
K8A,Q,S;
T9D;
T9D+L34F+A83E+Q149E+H193T+S332P+R340T;
I10V+D33E+M40L+A41T+Q67M+Y73F+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+T374A+S416A+D444Y+A469E+K470T+I473G+T517A+S522*;
I10V+F17S+D33E+M40L+A41T+Q67M+N72S+S76D+G78A+Q82K+Q137E+V219A+D249N+V272A+I337L+M356L+V397A+S416A+T421I+S424N+N441D+D444Y+V450I+K470T+I473S+V477I;
I10V+F17S+D33E+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+H164N+N168K+T172A+V219A+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+N415S+T421I+S424H+N441D+D444Y+S522P+P523V+V524E;
I10V+F17S+D33E+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+V272A+I337L+M356L+T374A+V397A+S416A+T421I+S424N+N441D+D444Y+V450I+A469E+K470T+I473G+T517A+S522P+P523V+V524E;
I10V+F17S+D33E+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+N168K+T172A+I222V+V228I+D249N+V272A+E333A+I337L+M356L+V397A+S416A+T421I+S424H+N441D+D444Y+A469E+K470T+I473S+V477I+E489A+A490V+T517A+S522*;
I10V+F17S+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+S269N+V272A+T320A+I337L+M356L+T374A+V397A+N415S+T421I+S424H+N441D+D444Y+A469E+K470T+I473S+V477I+T517A+S522P+P523V+V524E;
I10V+F17S+Q67M+N72S+S76D+G78A+Q82K+T104A+Q137E+N153K+R156Q+V219A+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+V397A+N415S+D420G+T421I+S424H+N441D+D444Y+V450I+A469E+K470T+I473G+T517A+S522*;
I10V+F17S+Q67M+N72S+S76D+G78A+Q82K+T92A+T104A+Q137E+R156Q+V159A+H164N+N168K+T172A+I222V+V228I+D249N+V272A;
I10V+F17S+Y53H+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+T172V+A177T+I222V+V228I+D249N+S269N+I337L+M356L+V397A+S416A+T421I+S424H+N441D+D444Y+A469E+K470T+I473G+T517A+S522*;
K13A+K129A;
K13A+Q68H+T92V+K118A+Q137E+R156Y+G200P;
K13A,R;
K18R;
R20A;
K21Q+K129A;
K21Q,R,T;
Q32H+M40L+R49G+D65E+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+T104A+Q137E+H164N+K202E+I222V+V228I+D249N+M356L+T374A;
D33V+Q68H+N168H+V450I;
L34F,I,M,V;
L34I+K129A;
D37G,N+K129A+R156Y;
E38I,V;
M40L+A41T+Q67M+N72S+S76D+G78A+Q82K+Q137E+N153K+H164N+D249N+V272A+I337L+M356L+V397A+N415S+T421I+S424N+N441D+V450I+E489A+A490V+T517A+S522*;
M40V;
L45I;
Q68H,M,N;
Q68H+G200P+N331F;
Q68H+K118A+K129A+R156Y+G200P+N331F;
Q68H+K118A+R156V+G200P+N331F;
Q68H+K118A+R156Y+H193T+D366H;
Q68H+K118R+R156F,Y;
Q68H+K118R+R156Y+G200P;
Q68H+K118S+R156F+G200P+G274D+N331F;
Q68H+K129A,T+R156K+G200P+N331F;
Q68H+R156F,V,Y+G200P+N331F;
Q68H+R156Y;
Q68H+R156Y+H193T;
Q68H+R156Y+H193T+D366H;
Q68H+R156Y+H193T+G200P+M310V;
Q68H+S76W+T92V+K118A+Q137E+R156Y+G200P+N331F;
Q68H+T92A,D,I,S,V,Y+K118A+K129A+R156Y+G200P+N331F;
Q68H+T92N+D97N+K118A+K129A+R156Y+G200P+N331F;
Q68H+T92S+K118A+K129A+R156Y+G200P+G274D+N331F;
Q68H+T92V+G200P+M310V;
Q68H+T92V+G200P+M310V+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+G200P+M310V+E446K;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+N331H,K,Q;
Q68H+T92V+K118A+K129A+R156Y+H193T;
Q68H+T92V+K118A+K129A+R156Y+H193T+D366H;
Q68H+T92V+K118A+K129A+R156Y+H193T+G200P+M310V;
Q68H+T92V+K118A+Q137E+N140F+R156Y+G200P+K470T;
Q68H+T92V+K118A+Q137E+R156Y+G200P+D324N;
Q68H+T92V+K118A+Q137E+R156Y+G200P+K470T;
Q68H+T92V+K118A+Q137E+R156Y+G200P+M310L;
Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118A,R+R156Y,F;
Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118R+R156Y+H193T+D366H;
Q68H+T92V+R156F+G200P+M310V+S484C;
Q68H+T92V+R156F,V,Y+G200P+M310V;
Q68H+T92V+R156F,V,Y+G200P+M310V+N331F;
Q68H+T92V+R156F,Y+H193T;
Q68H+T92V+R156F,Y+H193T+D366H;
Q68H+T92V+R156F,Y+H193T+G200P+M310V;
Q68H+T92V+R156Y;
S76E,I,K,M,R,T,V,W;
S76W+G200P;
S76W+G200P+A224P;
G78A+K118A++K129A+R156Y;
G78A+K118A+K129A+R156Y;
G78A+K118A+K129A+R156Y+G200P+N331F;
G78A+K118A+K129A+R156Y+K169A;
G78A,N,S;
G78A+T92V+K118A+K129A+R156Y;
G78A+T92V+K118A+K129A+R156Y+G200P+N331F;
G78A+T92V+K118A+K129A+R156Y+K169A;
L80V;
A83D,E,H,I,L,N,R,S,T,Y;
K87Q;
K87V+K129A+K169A;
T92I,V;
T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;
T92V+K118A+K129A+R156Y;
T92V+K118A+K129A+R156Y+G200P+N331F;
T92V+K118A+K129A+R156Y+H164N+G200P+N331F;
T92V+K129A+R156Y;
K101A+K129A;
K101R;
K101R+L102I;
T104A+P111Q+A117S+K129A+R156Y;
P111Q;
K118A+K129A;
K118A+K129A+F146L+R156Y+G200P+N331F;
K118A+K129A+Q137E+R156Y+G200P+N331F;
K118A+K129A+R156Y;
K118A+K129A+R156Y+A224P;
K118A+K129A+R156Y+G200P;
K118A+K129A+R156Y+G200P+M310V+N331F;
K118A+K129A+R156Y+G200P+N331F;
K118A+K129A+R156Y+G200P+N331F+N399I;
K118A+K129A+R156Y+K169A+G200P+N331F;
K118A+K129A+R156Y+K470T;
K118A,R;
K118A+R156Y;
K118A+R156Y+G200P;
D119L;
G120A;
S123P,T;
S123T+K129A+R156Y;
K129A,F,I,K,R,S,T;
K129A+K169A;
K129A+K176P;
K129A+K275Q;
K129A+K445S;
K129A+K470T;
K129A+Q137E+R156Y;
K129A+Q137E+R156Y+G200P;
K129A+Q137E+R156Y+K470T;
K129A+Q137E+V139K+N140F+Q147S+R156Y;
K129A+R156Y;
K129A+R156Y+A177T+V179I+A183S;
K129A+R156Y+A328G;
K129A+R156Y+D247G;
K129A+R156Y+D249G,N,S;
K129A+R156Y+D303I,K,S,V;
K129A+R156Y+D324N;
K129A+R156Y+D366H+T374A;
K129A+R156Y+D461N,Q,T;
K129A+R156Y+E288Q;
K129A+R156Y+G200P;
K129A+R156Y+G200P+G204T+R211K;
K129A+R156Y+H164N;
K129A+R156Y+H436Y;
K129A+R156Y+I10V+V14I+D19E;
K129A+R156Y+I222V+A224P+V228I+V232A;
K129A+R156Y+K176P,S;
K129A+R156Y+K275T;
K129A+R156Y+K322I+K454Q;
K129A+R156Y+K406N+N415G;
K129A+R156Y+K454Q;
K129A+R156Y+L380F+N383Y+D384G+N389T;
K129A+R156Y+N298F+E299N+G301T;
K129A+R156Y+N302K+D303L,S;
K129A+R156Y+N331F;
K129A+R156Y+P507A;
K129A+R156Y+R267H;
K129A+R156Y+R409L,T;
K129A+R156Y+S443D+K445S+L449I+V450I+S455N+M456Y;
K129A+R156Y+T244D;
K129A+R156Y+V159M+H164N+F165Y;
K129A+R156Y+V259I+R267K+L268K+S269A;
Q137D,E;
N140F;
K142A,Q,R;
F146C+H164C;
F146K,L;
F146L+K322I;
L148K+N168D;
Q149E;
R156A,D,E,F,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
R156Y+N331F;
V159M;
H164A,N;
L166I;
N168D;
K169A,Q,R;
K176P;
A177E,T;
K180R;
H193A,D,S,T;
R197A,L;
H199A;
G200A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
G200P+A224P;
K202N,Q,R;
S214E;
K217A;
A221K;
G225S;
V232A;
G237A,S,V;
K240A,Q,R;
K252A,Q,R;
G253A;
R267A;
L268I;
K275A,Q,R;
L278I;
F281L;
M290R;
R295A;
K306A,R;
K307Q;
M310I,L,V;
M310V+N399I;
R314A;
G316I;
K322A,R;
D324N;
N331A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y;
S332M,P;
S332P+V397I;
R340A,N,T;
K342A;
V345I;
K347A,Q,R;
D348G;
K353Q,R;
D366H;
M373Q;
T374A;
L380F;
K382A;
N383Y;
N389A,F,N,V;
W391V;
K392G,Q;
D395G;
G396P;
V397S;
N399I;
K406N;
G413A,S;
K414A;
N415S;
T417K;
F418I;
V431E;
H436A;
N441G+A442E+S443D;
S443E,K,Q;
K445A,R,S;
K445C+K470C;
H448A;
K454R;
S467R+G468S+A469T;
G468S,Y;
K470P,R,T;
I473T;
K476Q;
K482A,Q,R;
K488A,Q,R,T;
A490R;
G498A,D,S;
R500A,T,V;
H512A;
T517A+G518D;又は
G518D。
Q68H
S123P
R156Y
Q68H+R156Y
K129A+R156Y
S123T+K129A+R156Y
K129A+R156Y+G200P
Q68H+K118R+R156F
Q68H+R156Y+H193T
Q68H+R156F+G200P+N331F
Q68H+T92V+K118A+R156Y
K118A+K129A+R156Y+G200P+N331F
G78A+T92V+K118A+K129A+R156Y
Q68H+K129T+R156K+G200P+N331F
K118A+K129A+R156Y+K169A+G200P+N331F
T92V+K118A+K129A+R156Y+G200P+N331F
G78A+K118A+K129A+R156Y+G200P+N331F
G78A+T92V+K118A+K129A+R156Y+K169A
Q68H+T92V+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+Q137E+R156Y+N331F
Q68H+T92V+R156Y+G200P+M310V+N331F
Q68H+K118A+K129A+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+R156Y+G200P+N331F
Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+R156Y+H193T+D366H
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F
本発明はまた、本発明の変異体キシログルカナーゼ、又はキシログルカナーゼ活性を有するポリペプチドを含む組成物に関する。組成物はかかる変異体又はポリペプチドに濃縮される。用語「濃縮」とは、組成物のキシログルカナーゼ活性が増加して濃縮係数が1.1以上になることを指す。好ましくは、組成物は、薄い色、少ない臭気、及び許容可能な貯蔵安定性などの望ましい特徴を提供するために製剤化される。
本発明はまた、キシログルカナーゼ変異体の使用方法を目的とする。
本発明の洗濯洗剤組成物は、親キシログルカナーゼの単離変異体を含む。親キシログルカナーゼの単離変異体は、上記にてより詳細に説明がなされている。組成物は、好ましくは、特定の両親媒性アルコキシル化グリース洗浄ポリマーを含む。組成物は、好ましくは、洗浄性界面活性剤、好ましくは低濃度の洗浄性界面活性剤を含む。特定の両親媒性アルコキシル化グリース洗浄ポリマーについては、以下に詳しく記述される。洗浄性界面活性剤については、下記に詳しく記述される。組成物は、好ましくは、ランダムグラフトコポリマーを含む。好適なランダムグラフトコポリマーについては、以下に詳しく記述される。
本発明の一実施形態では、この組成物は固体洗濯洗剤組成物であり、好ましくは固体洗濯粉末洗剤組成物である。
本発明の両親媒性アルコキシル化グリース洗浄ポリマーは、布地及び表面からグリース粒子を欠失するように親水性と疎水性の特性が釣り合っている任意のアルコキシル化ポリマーを示す。本発明の両親媒性アルコキシル化グリース洗浄ポリマーの具体的な実施形態は、コア構造と、そのコア構造に結合した複数のアルコキシレート基と、を含む。
前記組成物は、洗浄性界面活性剤を含む。洗浄性界面活性剤は、アニオン性、非イオン性、カチオン性、及び/又は双性イオン性であり得る。好ましくは、この洗浄性界面活性剤はアニオン性である。この組成物は好ましくは2%〜50%、より好ましくは5%〜30%、最も好ましくは7%〜20%の洗浄性界面活性剤を含む。この組成物は2%〜6%の洗浄性界面活性剤を含み得る。この組成物は好ましくは、洗濯方法で洗浄液中100ppm〜5,000ppmの洗浄性界面活性剤を供給するような量の、洗浄性界面活性剤を含む。これは、洗濯方法で10g〜125gの液体洗濯洗剤組成物を洗浄液内に投入する場合に、特に好ましい。この組成物は水と接触して、典型的に、0.5g/L〜10g/Lの洗剤組成物を含む洗浄液を形成する。
ランダムグラフトコポリマーは、(i)不飽和C1〜C6カルボン酸、エーテル、アルコール、アルデヒド、ケトン、エステル、糖単位、アルコキシ単位、無水マレイン、飽和ポリアルコール(例えばグリセロールなど)、及びこれらの混合物からなる群から選択されるモノマーを含む親水性主鎖、並びに、(ii)C4〜C25アルキル基、ポリプロピレン、ポリブチレン、飽和C1〜C6モノカルボン酸のビニルエステル、アクリル酸又はメタクリル酸のC1〜C6アルキルエステル、及びこれらの混合物からなる群から選択される疎水性側鎖、を含む。
蛍光増白剤は、少なくとも何らかの可視光線を放出する。対照的に、布地色相剤は、可視光線スペクトルの少なくとも一部を吸収するため、表面の色合いを変えることができる。適切な布地色相剤としては、本明細書の試験方法の項に記載の試験方法1の要件を満たす染料、染料−粘土共役体、及び顔料が挙げられる。適切な染料としては、小分子染料及びポリマー染料が挙げられる。適切な小分子染料としては、ダイレクトブルー、ダイレクトレッド、ダイレクトバイオレット、アシッドブルー、アシッドレッド、アシッドバイオレット、ベーシックブルー、ベーシックバイオレット、及びベーシックレッドのカラーインデックス(C.I.)分類に該当する染料、又はこれらの混合物からなる群から選択される小分子染料が挙げられ、例えば次のものが挙げられる。
, UK))番号が、アシッドバイオレット17、ダイレクトブルー71、ダイレクトバイオレット51、ダイレクトブルー1、アシッドレッド88、アシッドレッド150、アシッドブルー29、アシッドブルー113、又はこれらの混合物からなる群から選択される小分子染料が挙げられる。
適切な補助剤物質としては、界面活性剤、ビルダー、キレート剤、移染防止剤、分散剤、追加の酵素及び酵素安定剤、触媒物質、漂白活性化剤、過酸化水素、過酸化水素源、予備形成済み過酸、ポリマー分散剤、粘土汚れ欠失/再付着防止剤、増白剤、泡抑制剤、染料、香料、構造伸縮性付与剤、柔軟仕上げ剤、キャリア、向水性物質、加工助剤、溶媒、及び/又は顔料が挙げられるが、これらに限らない。下記開示に加えて、このような他の補助剤の適した例、及び使用量は、米国特許第5,576,282号、同第6,306,812号及び同第6,326,348号に見ることができる。
本発明のキシログルカナーゼ変異体は、標準的な方法、手短に言えば、ランダムな及び/又は部位特異的な突然変異を遺伝子に導入し、バチルス・ズブチルス宿主細胞を変異遺伝子で形質転換し、形質転換された宿主細胞を発酵させ、発酵ブロスからキシログルカナーゼ変異体を得る、ことによって調製された。参照キシログルカナーゼ(配列番号3)を、同様の方法で、バチルス・ズブチルスにおいて組み換え的に産生した。
例えば精製で得た酵素サンプルのキシログルカナーゼ活性をAZCL−キシログルカンアッセイで測定した。
本発明のキシログルカナーゼ変異体の洗剤安定性を、液体洗剤中でのインキュベーション後の変異体の活性を測定することによって評価した。
実施例1に従って調製した酵素サンプルを、貯蔵安定性試験を開始する直前に解凍した。
洗剤の中で貯蔵された酵素サンプルの活性を以下の手段を用いて測定した。
1Mリン酸塩緩衝液pH7:
138グラムのNaH2PO4・H2Oを約750mLの水の中に溶解する。4N NaOHを加えてpH7.0とする。次に、最終容量を1000mLにする。
950mL水、50mL 1Mリン酸塩緩衝液pH7及び5mLのBerol 537(Akzo Nobelから供給される非イオン性界面活性剤)を混合する。最終pHを7.00±0.02に調節する。
Cellazyme Cタブレット、Megazyme International Ireland Ltdより供給、カタログ番号T−CCZ。タブレットは架橋染色HEセルロースを含有する。
アッセイ開始約30分前に、瓶を冷凍装置から約4℃の冷蔵庫に移した。アッセイ開始直前に瓶を冷蔵庫から取り出し、実験室の卓上の上に置いて開けた。
それぞれの酵素サンプルに関し、2つのAbs59測定値が存在する。
・−18℃で貯蔵されたサンプルのAbs590値であるA590f
・高温で貯蔵されたサンプルのAbs590値であるA590w
A590fからA590fを減算し(A590f−A590b)、A590wからA590fを減算する(A590w−A590b)。
%安定性=((A590w−A590b)/(A590f−A590b))×100%として計算された。
SIF=酵素サンプルの%安定性/親酵素(配列番号3)の%安定性
本実施例のキシログルカナーゼ変異体の洗剤安定性を、液体洗剤中でのインキュベーション後の変異体の活性を測定することによって評価した。
実施例1に従って調製した酵素サンプルを、貯蔵安定性試験を開始する直前に解凍した。
洗剤の中で貯蔵された酵素サンプルの活性を以下の手段を用いて室温で測定した。
パラ−ニトロフェノール−β−D−セロテトラオシド(pNP−β−D−セロテトラオシド)は、特定のキシログルカナーゼ酵素の触媒作用によって加水分解された合成基質である。
アッセイ緩衝液:100mM EPPS;0.01% Tween 20;pH8.0。
pNP−β−D−セロテトラオシド(CAS−#:129411−62−7;Toronto Research Chemicals;Canada)
基質溶液:アッセイ緩衝液中の1mM pNP−β−D−セロテトラオシド。
プレート1aは20μLのストレス負荷サンプルを収容し、プレート2は20μLのストレス無負荷サンプルを収容する。
ストレス負荷サンプルの酵素活性を同一のストレス無負荷サンプルの酵素活性で除したものとして%残留活性を計算した。
%残留活性=「Abs/分(ストレス負荷サンプル)」/「Abs/分(ストレス無負荷サンプル)」*100%。
下記は、布地洗濯に適した、本発明に従って製造された顆粒状洗剤組成物である。
2−NHにつき20個のエトキシル基を有するポリエチレンイミン(MW=600)。
3両親媒性グリース洗浄ポリマーは、−NHにつき24個のエトキシル基、及び−NHにつき16個のプロポキシル基を有するポリエチレンイミン(MW=600)である。
4次の構造を有する可逆性プロテアーゼ抑制剤。
Claims (13)
- 親キシログルカナーゼの単離変異体を含む洗剤組成物であって、前記変異体が、位置番号68、92、118、129、156、200、及び331からなる群から選択される3つ以上の位置の親キシログルカナーゼの改変を含み、この位置はアミノ酸配列番号3の位置に対応しており、
a)前記改変が、
i)前記位置を占める前記アミノ酸の下流へのアミノ酸の挿入、及び/又は
ii)前記位置を占める前記アミノ酸の欠失、及び/又は
iii)前記位置を占める前記アミノ酸の異なるアミノ酸での置換であり、
b)前記変異体が、配列番号3の前記親キシログルカナーゼのアミノ酸配列と85%同一性を有するアミノ酸配列を含み、かつ
c)前記変異体がキシログルカナーゼ活性を有する、組成物。 - 前記変異体が、以下の改変の組合せの1つ以上を含む、請求項1に記載の組成物。
K13A+Q68H+T92V+K118A+Q137E+R156Y+G200P;
Q68H+G200P+N331F;
Q68H+K118A+K129A+R156Y+G200P+N331F;
Q68H+K118A+R156V+G200P+N331F;
Q68H+K118A+R156Y+H193T+D366H;
Q68H+K118R+R156F,Y;
Q68H+K118R+R156Y+G200P;
Q68H+K118S+R156F+G200P+G274D+N331F;
Q68H+K129A,T+R156K+G200P+N331F;
Q68H+R156F,V,Y+G200P+N331F;
Q68H+R156Y+H193T+G200P+M310V;
Q68H+S76W+T92V+K118A+Q137E+R156Y+G200P+N331F;
Q68H+T92A,D,I,S,V,Y+K118A+K129A+R156Y+G200P+N331F;
Q68H+T92N+D97N+K118A+K129A+R156Y+G200P+N331F;
Q68H+T92S+K118A+K129A+R156Y+G200P+G274D+N331F;
Q68H+T92V+G200P+M310V;
Q68H+T92V+G200P+M310V+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+G200P+M310V+E446K;
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+N331H,K,Q;
Q68H+T92V+K118A+K129A+R156Y+H193T;
Q68H+T92V+K118A+K129A+R156Y+H193T+D366H;
Q68H+T92V+K118A+K129A+R156Y+H193T+G200P+M310V;
Q68H+T92V+K118A+Q137E+N140F+R156Y+G200P+K470T;
Q68H+T92V+K118A+Q137E+R156Y+G200P+D324N;
Q68H+T92V+K118A+Q137E+R156Y+G200P+K470T;
Q68H+T92V+K118A+Q137E+R156Y+G200P+M310L;
Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118A,R+R156Y,F;
Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F;
Q68H+T92V+K118R+R156Y+H193T+D366H;
Q68H+T92V+R156F+G200P+M310V+S484C;
Q68H+T92V+R156F,V,Y+G200P+M310V;
Q68H+T92V+R156F,V,Y+G200P+M310V+N331F;
Q68H+T92V+R156F,Y+H193T;
Q68H+T92V+R156F,Y+H193T+D366H;
Q68H+T92V+R156F,Y+H193T+G200P+M310V;
Q68H+T92V+R156Y;
G78A+K118A++K129A+R156Y;
G78A+K118A+K129A+R156Y;
G78A+K118A+K129A+R156Y+G200P+N331F;
G78A+K118A+K129A+R156Y+K169A;
G78A+T92V+K118A+K129A+R156Y;
G78A+T92V+K118A+K129A+R156Y+G200P+N331F;
G78A+T92V+K118A+K129A+R156Y+K169A;
T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;
T92V+K118A+K129A+R156Y;
T92V+K118A+K129A+R156Y+G200P+N331F;
T92V+K118A+K129A+R156Y+H164N+G200P+N331F;
T92V+K129A+R156Y;
K118A+K129A+F146L+R156Y+G200P+N331F;
K118A+K129A+Q137E+R156Y+G200P+N331F;
K118A+K129A+R156Y;
K118A+K129A+R156Y+A224P;
K118A+K129A+R156Y+G200P;
K118A+K129A+R156Y+G200P+M310V+N331F;
K118A+K129A+R156Y+G200P+N331F;
K118A+K129A+R156Y+G200P+N331F+N399I;
K118A+K129A+R156Y+K169A+G200P+N331F;
K118A+K129A+R156Y+K470T;
K118A+R156Y+G200P。 - 前記変異体が、アミノ酸配列番号3に対応する、位置123、位置137、位置193、又は位置76に対応する位置の1つ以上の改変をさらに含む、請求項1に記載の組成物。
- 前記変異体が、位置68の置換と、位置92、118、129、156、200及び331の2つ以上の置換とを含み、かつ前記変異体が、位置番号123、137、193、83、149、34、340、332、9、76、310、324、498、395及び366からなる群から選択される1つ以上の追加位置の1つ以上の置換を含む、請求項1に記載の組成物。
- 前記変異体が、位置156の置換と、位置68、92、118、129、200及び331の2つ以上の置換とを含み、かつ前記変異体が、位置番号10、13、14、19、37、68、78、123、137、139、140、147、159、164、165、169、176、177、179、183、204、211、222、224、244、247、249、259、267、268、269、275、288、299、301、302、303、310、324、328、366、380、383、384、389、406、409、415、436、443、445、449、450、454、455、456、461、470及び507からなる群から選択される1つ以上の追加位置の1つ以上の置換をさらに含む、請求項1に記載の組成物。
- 前記変異体が、位置129及び位置156の置換を含む、請求項5に記載の組成物。
- 前記変異体が、Q68H,N,L;R156Y,F,V,I,K,W,L,M;K118A,R;G200P,E,S,D;K129T,A,S;T92V,I,A,S;及びN331F,C;からなる群から選択される3つ以上の置換を含み、かつ前記変異体が、S123P,T;Q137E;H193T,S,D;A83E;Q149E;L34F,I,V;R340T,N;S332P;T9D;S76W,V,I,K,R,T;M310I,V,L;D324N;G498A,D;D395G及びD366Hからなる群から選択される1つ以上の追加位置の1つ以上の置換をさらに含む、請求項1に記載の組成物。
- 前記変異体が、以下の置換の組合せの1つ以上を含む、請求項1に記載の組成物。
K129A+R156Y+G200P
Q68H+K118R+R156F
Q68H+R156F+G200P+N331F
Q68H+T92V+K118A+R156Y
K118A+K129A+R156Y+G200P+N331F
G78A+T92V+K118A+K129A+R156Y
Q68H+K129T+R156K+G200P+N331F
K118A+K129A+R156Y+K169A+G200P+N331F
T92V+K118A+K129A+R156Y+G200P+N331F
G78A+K118A+K129A+R156Y+G200P+N331F
G78A+T92V+K118A+K129A+R156Y+K169A
Q68H+T92V+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+Q137E+R156Y+N331F
Q68H+T92V+R156Y+G200P+M310V+N331F
Q68H+K118A+K129A+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+R156Y+G200P+N331F
Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+R156Y+H193T+D366H
Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F
Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F - 前記組成物が液体洗濯洗剤組成物である、請求項1に記載の組成物。
- 前記組成物が、
(a)両親媒性アルコキシル化グリース洗浄ポリマー;
(b)ランダムグラフトコポリマー、ここで前記ランダムグラフトコポリマーは、
(i)不飽和C1〜C6カルボン酸、エーテル、アルコール、アルデヒド、ケトン、エステル、糖単位、アルコキシ単位、無水マレイン酸、グリセロールなどの飽和ポリアルコール、及びこれらの混合物からなる群から選択されるモノマーを含む親水性主鎖と、
(ii)C4〜C25アルキル基、ポリプロピレン、ポリブチレン、飽和C1〜C6モノカルボン酸のビニルエステル、アクリル酸又はメタクリル酸のC1〜C6アルキルエステル、及びこれらの混合物からなる群から選択される疎水性側鎖と、を含み;
(c)次の一般構造を有する化合物:ビス((C2H5O)(C2H4O)n)(CH3)−N+−CxH2x−N+−(CH3)−ビス((C2H5O)(C2H4O)n)(式中、nは20〜30であり、及びxは3〜8である。)又はその硫酸化若しくはスルホン酸化変異体;
から選択される1つ以上の成分を含む、請求項1に記載の組成物。 - 前記組成物が香料マイクロカプセルを含む、請求項1に記載の組成物。
- 前記組成物が布地色相剤を含む、請求項1に記載の組成物。
- 前記組成物が固体形状である、請求項1に記載の組成物。
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CA2724699A1 (en) | 2009-12-10 |
EP3404088A1 (en) | 2018-11-21 |
EP2300588A1 (en) | 2011-03-30 |
JP2011524438A (ja) | 2011-09-01 |
RU2525669C2 (ru) | 2014-08-20 |
CA2724699C (en) | 2015-02-10 |
US20090312221A1 (en) | 2009-12-17 |
AR072058A1 (es) | 2010-08-04 |
MY159940A (en) | 2017-02-15 |
ZA201008419B (en) | 2019-06-26 |
WO2009148983A1 (en) | 2009-12-10 |
BRPI0913570A2 (pt) | 2015-12-15 |
CN102057030B (zh) | 2016-05-11 |
RU2010148557A (ru) | 2012-07-27 |
US8202831B2 (en) | 2012-06-19 |
EP2300588B1 (en) | 2019-02-06 |
MX2010013276A (es) | 2010-12-21 |
CN102057030A (zh) | 2011-05-11 |
ES2720369T3 (es) | 2019-07-19 |
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